Fautsch, Michael P.

Aims To determine the associations between measures of oestrogen exposure and Fuchs endothelial corneal dystrophy (FECD) severity. Methods Clinic-based cross-sectional study of 32 postmenopausal women and 11 men with mild or severe FECD, age>55 years. Participants completed questionnaires for data on demographics, anthropometric factors, medical history and potential risk factors for FECD. Women completed an additional reproductive history questionnaire used to calculate the months of lifetime oestrogen exposure. Slit-lamp biomicroscopy, specular microscopy, corneal Scheimpflug tomography and laboratory testing ( TCF4 repeat expansion quantification, total estradiol, free estradiol, sex hormone binding globulin (SHBG), calcaneal bone density) were performed. Logistic regression models were developed to predict FECD severity based on three-way interactions of each oestrogen exposure measure, sex and TCF4 genotype. Results 43 patients were enrolled in the study (mild FECD: 17 women, 3 men; severe FECD: 15 women, 8 men). Serum-free estradiol was higher in the severe compared with mild FECD group (0.21±0.2 vs 0.09±0.1 pg/mL; p=0.046). When stratified by sex, men showed no significant associations between oestrogen measures and FECD severity. However, in women, the odds of severe FECD were increased with more months of lifetime oestrogen exposure (all women; log OR (95% credible interval): 1.3 (0.14 to 4.3)), higher concentrations of free estradiol (all women; 2.1 (0.0049 to 10)), greater % free estradiol (only women without TCF4 repeat expansion; 1.3 (0.16 to 3.8)) and higher concentrations of SHBG (only women with TCF4 repeat expansion; 2.2 (0.45 to 9.1)). Conclusions While the application of these data is constrained by the limited number of participants, a clinic-based sample, small number of men compared with women and single-point measures of serum hormone measures, these data suggest that FECD severity is associated with higher oestrogen exposures in women but not men and may be dependent on TCF4 repeat expansion status.

To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 (EGR1) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P < 0.01; MEL624 27%, P < 0.001) and conjunctival (YUARGE 13-3064 33%, P < 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P < 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P = 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P = 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.

AbstractGlaucoma, a progressive neurodegenerative ocular disease, is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is the most common—and the only treatable—risk factor for glaucoma. IOP is generated by the balance between production and removal of aqueous humor in the anterior part of the eye, and the latter happens either through the uveoscleral or the conventional pathway. Although both pathways are important for aqueous humor removal, dysfunction within the conventional pathway is more commonly associated with increased resistance leading to elevated IOP and glaucoma. The conventional pathway can be separated into proximal (trabecular meshwork and inner wall of Schlemm's canal) and distal (outer wall of Schlemm's canal, collector channels, and episcleral vasculature) regions. Both regions contribute resistance to aqueous humor removal, but the proximal region has been studied more extensively due to the availability of model systems. In contrast, little is known about the role of the distal region in outflow resistance, largely due to the lack of suitable in vitro models. To address this, we have developed a novel method of isolating and culturing vascular distal outflow pathway (VDOP) cells from the distal outflow region of human eyes. VDOP cells can be used to study the physiological and molecular functions of cells in the distal outflow region and can help in the development of ocular hypotensive drugs that specifically target this area. We also provide a protocol describing immunohistochemical methods to validate the molecular profile of these cells, utilizing cell surface markers that distinguish them from adjacent cells. © 2022 Wiley Periodicals LLC.Basic Protocol 1: Isolation and culture of VDOP cellsBasic Protocol 2: Analysis of cell surface markers